Light activated gene transduction for cell targeted gene delivery in the spinal column

ABSTRACT

In accordance with the present invention, methods and structures are provided for the treatment of functional spinal unit injuries through the use of light activated gene therapy to induce bone fusion through the introduction of a desired gene into a patient&#39;s spinal tissue. Methods and structures are also provided for the utilization of ultraviolet light activated gene therapy to repair/rebuild an injured intervertebral disc through the introduction of a desired gene into a patient&#39;s spinal tissue. An implant system including a light probe and an implant with which r-AAV is integrated is also provided.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.10/357,273, filed Jan. 31, 2003, which is incorporated herein byreference in its entirety.

GOVERNMENT INTEREST

This invention was made with Government support under NIH Contract#AR45972, an RO1 grant awarded by NIAMS. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of gene therapy. According tothe present invention, methods are provided for the treatment offunctional spinal unit injuries through the use of light activated genetherapy to introduce a desired gene into a patient's tissue. Anembodiment of the present invention includes methods for the utilizationof light activated gene therapy to repair/rebuild an injuredintervertebral disc. Alternate embodiment provide an implant systemhaving UV activated viral vector integrated with an implant.

2. Description of the Related Art

Currently, treatment for injured spines often involves “fusion” orinciting the biological union of bones by inserting bone grafts ordevices within the functional spinal units (FSU), e.g., between twovertebra. In addition, the effective manipulation of certainosteobiologic molecules via gene therapy can be used to incite bonefusion within a functional spinal unit (FSU). A FSU is composed of twovertebra, a nearby nerve root, and a human interverterbral disc betweenthe two vertebra. This disc, which cushions shock to the spine and lendsstability to the FSU, is composed of water, collagen (Type I and II),and glycosaminoglycans (GAG).

An aging or degenerate disc is often characterized by reduced water,increased Type I collagen, decreased Type II collagen, and decreasedGAG. This aging or degenerating, which is incompletely understood,generally results in decreased biomechanical shock absorption, increasedrange of motion and pain and/or disability.

Somatic cell gene therapy is a form of treatment in which the geneticmaterial of a target cell is altered through the administration ofnucleic acid, typically in the form of DNA. In pursuit of effective invivo administration routes, scientists have harnessed the otherwisepotentially deleterious ability of viruses to invade a target cell and“reprogram” the cell through the insertion of viral DNA. Byencapsulating desirable genetic material in a viral particle, or“vector,” minus some of the viral DNA, the effective and targeteddelivery of genetic material in vivo is possible. As applied to spinalspecific treatments, gene therapy offers the ability to make use ofosteobiological molecules, including both intracellular andextracellular proteins, to incite bone fusion and/or disc repair.

In particular, the desirable qualities of adeno-associated viruses (AAV)have led to further study of potential gene therapy uses. As a vehiclefor gene therapy recombinant forms of AAV, or r-AAV, offer manyadvantages including the vector's ability to infect non-dividing cells(e.g., chondrocytes, or cells within cartilage), the sustained targetgene expression, the low immune response to the vector, and the abilityto transduce a large variety of tissues. The AAV contains a singlestrand DNA (ssDNA) genome. Under normal conditions, AAV is present inhumans in a replication incompetent form, due to the fact the AAV alonedoes not encode the enzyme required for replication of the second DNAstrand. Successful r-AAV transduction often requires the presence of aco-infection with an adenovirus or the exposure of the host cell to DNAdamaging agents, such as γ-irradiation. The introduction of either theco-infection or the DNA damaging agents dramatically induces the ratelimiting step of second strand synthesis, i.e. the second strand of DNAwhich is synthesized based on the vector inserted first strand. However,making use of these DNA damaging agents is impractical because theadministration of an adenovirus co-infection to a patient is notpractical or desirable and the site specific and safety issues involvedwith using γ-irradiation are undesirable as well.

In the past, attempts have been made to induce r-AAV transduction invitro using UV radiation having a wavelength of 254 nm. Unfortunately,no effective therapeutic method or apparatus was developed based onthese experiments due to the long exposure times involved with using 254nm UV radiation, the difficulties of delivering 254 nm UV radiation to asurgical target site, and the inability to position the 254 nm UV lightsource so as to allow effective penetration of a target cell.

SUMMARY OF THE INVENTION

Preferred embodiments of the present invention provide structures andmethods for treating a patient's spine using light activated genetherapy.

In accordance with an embodiment of the present invention, a method ofintroducing a desired gene into a patient's spinal tissue is provided. Alight probe is locating proximate to target cells. The transduction ofthe ultraviolet light activated viral vector is activating by locallyadministering ultraviolet light to spinal target cells using the lightprobe. The ultraviolet light activated viral vector is deliveredproximate to the spinal target cells.

In accordance with another embodiment of the present invention, a methodof introducing a desired gene into a patient's spinal tissue is providedincluding inserting an implant proximate to spinal target cells withinthe patients spinal tissue. A light probe is located proximate to spinaltarget cells. The transduction of a ultraviolet light activated viralvector is activated by locally administering ultraviolet light to spinaltarget cells using the light probe. The ultraviolet light activatedviral vector is delivered proximate to the spinal target cells.

In accordance with yet another embodiment of the present invention, aspinal implant system for introducing a desired gene into a patient'stissue is provided. The system includes an expandable implant configuredto be inserted into a patient's spine in a minimally intrusive surgicalprocedure and an ultraviolet activated viral vector integrated with theexpandable spacer.

In accordance with still another embodiment of the present invention, agene therapy system for increasing the transduction of an ultravioletlight activated viral vector in a patient's spinal tissue is provided.The gene therapy system includes a power source which powers a lightsource producing an ultraviolet light beam. An optical coupler is alsoincluded for guiding the light beam into a light delivery cable. A timedshutter, having a user interface for controlling the shutter, is locatedin line with the light beam. A light probe, which receives the lightbeam from the light delivery cable, is configured to output the lightbeam proximate to the target cells in a patient's spine in order toincrease the transduction of the ultraviolet light activated viralvector. An optical connector connects the light delivery cable to thelight probe.

A feature of preferred embodiments of the present invention the abilityto overcome the problems involved with using traditional UV andγ-irradiation, by using locally administered UV, preferably longwavelength UV light, in order to induce the target cells to moreeffectively stimulate the transduction of a UV activated viral vector,such as recombinant adeno-associated virus r-AAV. In certain preferredembodiments, the desirable genetic material carried in the UV activatedviral vector is then able to facilitate the rebuilding of importantcomponents of the FSU, such as GAG and collagen, or to effect boneformation resulting in fusion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is flowchart of a method of treating a patient's spinal tissueusing a UV light activated viral vector and a UV light probe, inaccordance with an embodiment of the present invention.

FIG. 2A is a side view schematic of the light source and user interfacecomponents of a light probe system, in accordance with anotherembodiment of the present invention.

FIGS. 2B is a schematic of light probe forming part of the light probesystem shown in FIG. 2.

FIG. 3 is a schematic of a syringe for introducing a UV activated vectorinto a patient's spinal tissue.

FIG. 4A-D are perspective schematics of example collapsible spinalimplants, in accordance with yet another embodiment of the presentinvention.

FIG. 4E is a cross section schematic of the expanded implant of FIG. 4D,the expanded implant shown located between two vertebra.

FIG. 5 is flowchart of a method of treating a patient's spinal tissueincluding a spinal implant, in accordance with a further embodiment ofthe present invention.

FIGS. 6-8 are graphs of the results of the proof of principle experimentof Example 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The term “AAV” refers to adeno-associated virus, while “r-AAV” refers torecombinant adeno-associated virus. Preferably, r-AAV includes only agene, which is desired to be introduced into the patient's spinaltissue, and the flanking AAV inverted terminal repeats (ITR's) thatserve as the packaging signals.

“Ultraviolet radiation” and “ultraviolet light,” also known as “UV”,refer to the portions of the electromagnetic spectrum which havewavelengths shorter than visible light. The range of wavelengthsconsidered to be ultraviolet radiation, from about 4 nanometers to about400 nanometers, is further subdivided into three subgroups, UVA, UVB,and UVC. “UVA” is the portion of ultraviolet radiation which includeswavelengths from 320 nm up to and including 400 nm. “UVB” is the portionof ultraviolet radiation which includes wavelengths from 280 nm up toand including 320 nm. “UVC” is the portion of ultraviolet radiationhaving a wavelength less than 280 nm.

The term “long wavelength UV” refers to ultraviolet radiation or lighthaving a wavelength greater than or equal to 255 nm, but not more than400 nm.

A “viral vector” refers to a virus, or recombinant thereof, capable ofencapsulating desirable genetic material and transferring andintegrating the desirable genetic material into a target cell, thusenabling the effective and targeted delivery of genetic material both exvivo and in vivo. A “UV activated viral vector” or “UV light activatedviral vector” is any virus, or recombinant thereof, whose replication isregulated by ultraviolet light. Recombinant adeno-associated virus(r-AAV) is included in the group of viruses labeled UV activated viralvectors.

The term “LAGT” refers to light activated gene transduction, while “LAGTprobe” or “light probe” or “long UV wavelength light probe” refers tothe medical device which delivers ultraviolet light to the target siteand effectuates the transduction of the desired gene carried by thevector.

“Implant” or “spacer implant” is any structure designed to be insertedproximate to the spine for the purpose of aiding the treatment of thespinal target site where the implant is inserted.

FIG. 1 shows a method of treating a patient's spinal tissue. A lightprobe is also located 100 proximate to target cells in a patient'sspinal tissue. The transduction of an ultraviolet (UV) activated viralvector is then activated 110 by locally administering ultraviolet lightto the target cells using the light probe. The ultraviolet (UV)activated viral vector, containing a desired gene, is delivered 120proximate to the target cells.

It should be noted that the method of FIG. 1 may be performed in otherpreferred embodiments in a different order than the textually outlinedabove. For example, in another preferred embodiment the vector isdelivered prior to locally administering the ultraviolet light.

In one preferred embodiment the LAGT probe activates a UV activatedviral vector transduction, including r-AAV transduction, by radiatingthe viral vector infected target cell with locally administered UVradiation, while in a preferred alternate embodiment the target cell isactivated with locally administered long wavelength UV radiation havinga wavelength specifically from 255 nm up to and including 400 nm. Inother preferred embodiments, the wavelength of the UV light ranges fromabout 280 to about 330. More preferably, the locally administered UVradiation has a wavelength from 315 nm to 355 nm, most preferably about325 nm. In an alternate embodiment the ultraviolet radiation has awavelength of about 4 nm to about 400 nm, while in another alternateembodiment the ultraviolet radiation has a wavelength of 290 nm.

FIGS. 2A and 2B illustrate separate components of a UV radiationdelivery system, with FIG. 2A showing the UV light generator 10, userinterface system, and FIG. 2B showing the light probe 26. Note the lightprobe 26 is operatively connected to the UV light generator 10 by thelight delivery cable 24.

With reference to FIG. 2A, the UV radiation delivery system includes alight source 12 with the desired wavelength UV output. In addition, anoptical coupler 14 transmits the light from the light source 12 into alight delivery cable 24, such as an optical fiber cable or bundle, thattransmits the light to the target site via a light probe 26 (FIG. 2B). Atimed shutter 16 is located in the path of the light beam between thelight source 12 and the optical coupler 14 in order to control thelength of time the patient is exposed to UV light via the light probe 26(FIG. 2B). The timed shutter 16 is operatively connected via connectors22 to a shutter controller 18 and a shutter control interface 20.

FIG. 2B shows a light probe 26 as a component of UV radiation deliverysystem for use with the component, shown in FIG. 2. The light probe 26is configured to locally irradiate a target cells infected by a UVactivated viral vector. The light probe 26 fiber-optically transmit anappropriate wavelength light, which originates from the light source 12(FIG. 2A), through a light guide 30 to a light guide terminator 34configured to irradiate target cells with UV light and thereby“activate” r-AAV transduction in the target cells. The light guide 30 ispreferably surrounded by a housing 32. The light guide terminator 34,e.g., a microlens or cylindrical diffusing lens, is preferablyconfigured to allow the effective irradiation of the desired targetcells. The light probe 26 is preferably both shaped in the form of anarthroscope and interchangeable with alternate light probes having adiffering configurations. For example, the light probe can be configuredto have different forms in order to more effectively access differenttreatment sites. Preferably, the optical connector 28 also allows thelight probe 26 to be selectively detached from the light delivery cable24 when desired. The light probe 26 is also preferably configured to besterile and disposable. In certain alternate embodiments, the UVradiation delivery system also includes a targeting laser beam to enableaccurate delivery of the light. Standard surgery tools as recognized bythose skilled in the art, for example cannulas and trochars, may also beincorporated into the disclosed method.

In the embodiment shown in FIG. 2A, the light source is contained withina housing, while in certain alternate embodiments the light source isoperatively joined to the housing. It should be understood that theexact shape and size of the light probe 26 shown in FIG. 2A, andespecially the light probe tip, will vary depending on the particularapplication and target site as would be understood by one skilled in theart. For example, the light probe 26 can be configured to access anintervertabral disc in a patient's spine or the cartilage in a patient'sjoint. The preferred embodiments include a light source comprising alaser tuned to the appropriate long UV wavelength. In preferredembodiments, the UV radiation delivery system, whether it be a lamp orlaser based system, will be optimized based on considerations such ascost and technical simplicity. In addition, the UV radiation deliverysystem can also include a targeting laser beam to enable accuratedelivery of the light. Standard surgery tools, for example cannulas andtrochars, may also be used. In accordance with alternate embodiments,the light probe could be designed for external use, such as irradiatingan implant having r-AAV integrated therewith, outside of the patient andsubsequently inserting the implant to a spinal target site.

Alternate embodiments of the UV radiation delivery system employ as alight source, a lamp, such as a high intensity argon lamp. In thesealternate embodiments, the UV radiation delivery system further includesa wavelength selecting device, such as a dichroic mirror and/or opticalfilter, set to transmit the desired wavelength UV light and rejectunwanted light wavelengths.

As shown in FIG. 3, an injecting device 36 having a housing 38 and aplunger mechanism 40 is preferably employed in conjunction with the UVradiation delivery system of FIGS. 2A and 2B. Preferably, the injectingdevice 36 is configured for delivering a UV activated viral vector, suchas r-AAV, to the target site using minimally invasive surgicaltechniques. In alternate preferred embodiments, the injecting device canbe configured to inject an implant to a target site in a patient.

Surgery tools, other than the injecting device 36 shown in FIG. 3, whichcan be involved in certain preferred embodiments include a cannula, atrochar and a power percutaneous disc resector, (all not shown) whichincreases space within a disc space. It should be noted that the sizeand design of these tools would vary to adapt to both the treatment goaland the target site. Alternate embodiments of these tools would bedesigned to access cervical, thoracic, and lumbar disk spaces, as wellas the facet joints.

Referring to FIGS. 4A-4E, alternate preferred embodiments provide aspinal implant system, including “spacer” implants which createtemporary mechanical rigidity between discs while the target cellsrespond to the introduction of the desired gene into the patient'stissue. These solid platforms are preferably expandable and designed assurgical implants. These carefully engineered solid platforms orimplants can also be formed in a number of shapes, including but notlimited to an unfolding geodosic dome 42 or tetrahedrons (not shown),umbrella/dome (not shown), an expanding cylinder 44, and springs whichuncoil to increase diameter. Expanding cylinder 44 is shown in acompacted shape in FIG. 4A and an expanded state in FIG. 4B (and alsoFIG. 4E), while unfolding geodosic dome 42 is shown in a compacted shapein FIG. 4C and an expanded state in FIG. 4D. Preferably, these implantsare produced with implant integrated UV activated viral vector. Forexample, r-AAV can be integrated with the implant through bonding orcoating the r-AAV to the implant, absorbing the r-AAV into the implant,and/or baking the r-AAV to the implant surface. In alternate preferredembodiments the implant is delivered to a target site separate from theUV activated viral vector. Non-limiting examples of solid platforms withwhich UV activated viral vectors could be integrated include spinalspacers, as shown in FIG. 6E, and also other spinal surgical implants.

FIG. 4E shows the expanding cylinder 44 of FIG. 4A and 4B in an expandedstate, to which a UV activated viral vector is preferably integrated,placed between two vertebra 50 in order to facilitate the rebuilding orrepair of the intervertebral disc 48. In another embodiments the implantis delivered to a target site separate from the UV activated viralvector.

It should be understood that structural support implants incorporatingsuch conventional structures as, for example, but not limited to,plates, rods, wire, cables, hooks, screws, are also advantageouslyuseful with preferred embodiments provided herein. The support structuremay be formed from material such as, but not limited to, metal,carbon-fiber, plastic, and/or reabsorbable material.

In another preferred embodiment, shown in FIG. 5, a implant is inserted200 at a spinal target site, preferably using a minimally invasive routesuch as a stab incision. Target cells are infected 210 with a UVactivated viral vector, such as r-AAV, containing a desired gene,preferably by attaching the vector to the implant prior to insertion.For example, r-AAV can be integrated with the implant through coating orbonding the r-AAV to the implant, absorbing the r-AAV into the implant,and/or baking the r-AAV to the implant surface. A light probe is placedor located 220 proximate to the target cells. The light probe activates230 the infected target cell's UV activated viral vector transduction.In an alternative embodiment, the vector is delivered to the target sitein a step separate from the insertion of the implant.

It should be noted that the method of FIG. 5, and the other methodsprovided herein, may depending on the desired order and outcome, beperformed in other preferred embodiments in a different order than thetextually outlined herein.

In certain preferred embodiments, the spacer implants can be used toreconstitute disk height and preserve FSU geometry while the surroundingbone fusion progresses. In these embodiments, the UV activated viralvector includes bone forming genes. The light probe would then directlyactivate target cells to transduct the viral vector.

In other embodiments employing spinal implants, the spacer implants canbe used to reconstitute disk height and preserve FSU geometry while theinvertebral disc regenerates to form a repaired or rejuvenatedinvertebral disc. In another preferred embodiment, structural supportimplants can be used to reconstitute disk height and preserve FSUgeometry while the invertebral disc regenerates to form a repaired orrejuvenated invertebral disc. In these embodiments, the UV activatedviral vector includes disc regenerating genes. In certain preferredembodiments, these structural support implants would be attached tobone, e.g., a plate screwed into the spinal column. The light probewould then directly activate target cells to transduct the viral vector.

In one embodiment the LAGT probe can activate the r-AAV transduction byirradiating the target cell with UV radiation, while in an alternateembodiment the target cell is activated with long wavelength UVradiation. Preferably, the injected r-AAV can carry bone forming genesand/or disc regenerating genes depending on the desired treatment goal.In addition, a spacer implant, in combination with bone forming genes,may be inserted between two vertebra in order to facilitatereconstituted disc height and bone fusion.

Embodiments of the present invention include both in vivo and ex vivoapplications. In the ex vivo application the radiation dose would beapplied to cells or biological material external to the patient and thendelivered, preferably through injection, to the desired site oftreatment. In the in vivo application the LAGT probe and the UVactivated viral vector are preferably introduced to the treatment siteusing minimally invasive surgical techniques, such as stab incisions.Alternate in vivo embodiments employ direct visualization surgicaltechniques.

A UV light activated viral vector or UV activated vector is any virus,or recombinant thereof, whose replication is regulated by ultravioletlight. Preferred embodiments of UV activated viral vectors are viruseswith single stranded DNA, the virus being capable of allowing atherapeutically significant increase in virus transduction when a virusinfected target cell is exposed to a therapeutic dose of ultravioletradiation. More preferred embodiments include UV activated viral vectorscapable of infecting non-dividing cells, effectuating sustained targetgene expression, eliciting a low immune response to the vector, andpossessing an ability to transduce a large variety of tissues. Mostpreferably, the UV light activated vector is r-AAV.

Proof of principle experiments, both ex vivo and in vivo based, arecurrently under way to determine the optimal wavelengths for activatingthe gene therapy. The determination of more preferred wavelengths isbased on among other factors, the ability to effectively penetrate atarget cell, ease and efficiency of fiber optic transmission, theability to trigger the transduction of a UV activated vector (such asr-AAV), and the length of time a patient must be exposed to receive atherapeutic dose of ultraviolet radiation. Preferably, the LAGT systemdelivers long wavelength ultraviolet radiation in the range of 315 nm to400 nm. Current experiments support the use of ultraviolet radiationhaving a wavelength from 315 nm to 355 nm, but it is believed that theseexperiments will ultimately support ultraviolet radiation having awavelength from 315 to 400 nm. In addition, alternate embodiments employa laser which produces ultraviolet radiation having a wavelength ofabout 290 nm. Once specific wavelengths are determined, the disclosedcomponents can be optimized for these specific wavelengths.

The wavelength of the ultraviolet light generated in order to activateUV activated viral vector transduction, including r-AAV transduction, intarget cells is preferably 255, 256, 258, 265, 275, 285, 290, 295, 305,314, 325, 335, 345, 355, 365, 375, 385, 395, or 400 nanometers. Morepreferably, the wavelength of the ultraviolet light is 290, 295, 300,305, 310, 315, 316, 317, 322, 325, 327, 332, 337, 342, 347, 352, 357,362, 367, 372, 377, 382, 387, 392, 393, 394, 395, 396, 397, 398, or 399nanometers. Most preferably, the wavelength of the ultraviolet light isabout 325 nanometers.

Table 1 shows example growth factors, signaling molecules andtranscription factors which genes inserted into a viral vector couldencode for in the practice of osteo-integration and spine fusion. Incertain preferred embodiments, genes encoding these molecules andfactors are integrated with implants. The list contained in Table 1 isprovided for illustrative purposes and should not be taken as limitingthe embodiments of the invention in any way.

Table 2 shows example molecules which genes inserted into a viral vectorcould encode for in the practice of periprosthetic osteolysis. Incertain preferred embodiments, genes encoding these molecules areintegrated with implants. The list contained in Table 2 is provided forillustrative purposes and should not be taken as limiting theembodiments of the invention in any way. TABLE 1 osteo-integration andspine fusion: (a) GROWTH FACTORS Transforming Growth Factor beta (TGFb)1, 2 and 3 bone morphogenetic protein (BMP) 1, 2, 4, 6 and 7 parathyroidhormone (PTH) parathyroid hormone related peptide (PTHrP) fibroblastgrowth factor (FGF) 1, 2 insulin-like growth factor (IGF) (b) SIGNALINGMOLECULES AND TRANSCRIPTION FACTORS LMP-1 Smad 1, 5, 8 dominant-negativeSmad 2, 3 Smurf2 Sox-9 CBFA-1 ATF2

TABLE 2 perioprosthetic osteolysis: soluble tumor necrosis factorreceptors TNFR, TNFR:Fc osteoprotegerin (OPG) interleukin-1 receptorantagonist (IL-1RA), IL-1RII:Fc interleukin-4,10 and viral IL-10

The results of a completed proof of principle experiment are shown inExample 1.

EXAMPLE 1

I. Methods

A. Isolation of Human Mesenchymal Stem Cells

Human Mesenchymal Stem Cells (hMSC) were isolated from patient bloodsamples harvested from the iliac crest. The blood samples were dilutedin an equal volume of sterile Phosphate Buffered Saline (PBS). Thediluted sample was then gently layered over 10 ml of Lymphoprep (MediaPrep) in a 50 ml conical tube (Corning). The samples were thencentrifuged at 1800 rpm for 30 minutes. This isolation protocol is astandard laboratory technique, and the resulting gradient that formedenabled the isolation of the hMSCs from the layer immediately above theLymphoprep. The isolated fraction was placed into a new 50 ml conicaltube, along with an additional 20 ml of sterile PBS. The sample wascentrifuged at 1400 rpm for 8 minutes. The supernatant was removed thecell pellet was resuspended in 20 ml for fresh PBS, and centrifugedagain for 8 minutes at 1400 rpm. Afterwards the supernatant was removed,the cell pellet was resuspended in 10 ml of Dulbecco's Modified EagleMedium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1%Penicillin/Streptomycin (P/S) (Invitrogen). The hMSCs were grown andpassed as necessary in a 37°/5% CO₂, water-jacketed incubator (FormaScientific).

B. 325 nm UV treatment of Human Mesenchymal Stem Cells

Prior to irradiation, hMSCs were plated at a density of 5×10⁴ cells/wellin 12-well plates. The cells were allowed to sit down overnight. Thenext morning the media was removed immediately prior to irradiation. Thecells were irradiated at various doses (500 J/m², 1000 J/m², 3000 J/m²,6000 J/m², or 10,000 J/m²) of 325 nm UV light using cadmium laser system(Melles Griot). After irradiation, fresh media, either with or withoutrecombinant adeno-associated virus was added to the wells.

C. Infection of Human Mesenchymal Stem Cells with RecombinantAdeno-Associated Virus

Infections were carried out in 12-well dishes. The cells were infectedat various multiplicities of infection (MOIs=10, 100, and 1000), using arecombinant adeno-associated virus carrying the bacterialβ-galactosidase reporter gene (rAAV-LacZ via UNC-Chapel Hill GeneTherapy Vector Core Facility). After being irradiated, the cells wereinfected with the predetermined amount of virus in a total volume of 500μl of DMEM/10% FBS/1% P/S. Two hours after the initial infection, andadditional 1 ml of media was added to the cultures. The cultures werethen allowed to incubate (37°/5% CO₂) for forty-eight hours beforeharvest for analysis.

D. Quantifying Recombinant Gene Expression

Forty-eight hours after infection, the cells were harvested; celllysates were made and analyzed using a commercially availableLuminescent β-gal Reporter System. (BD Biosciences), Briefly,experimental cell samples were removed from the 12-well dish using 0.25%Trypsin-EDTA. The cell suspension was transferred to a 1.5 ml conicaltube and the cells were pelleted via a 15 second centrifugation at13,000 rpm. The cell pellet was washed using two successive rounds ofresuspension in ice cold PBS and pelleting for 15 seconds at 13,000 rpm.The final pellet was resuspended in 75 μl of Lysis Buffer (100 mMK₂HPO₄, 100 mM KH₂PO₄, 1 M DTT) and subjected to three rounds offreeze/thaw in an isopropanol dry ice bath and a 37° water bath. Thelysates were centrifuged for a final time for 5 minutes at 13,000 rpm.Aliquots (15 μl) of the resulting supernatant were incubated with theprovided substrate/buffer solution for one hour and then analyzed usinga standard tube luminometer. The read out of this analysis is expressedin Relative Light Units (RLU) in the Results section.

II. Results

A. Exposure to 325 nm UV Increased the Level of Reporter Gene Expression

Exposure to 325 nm UV prior to infection with rAAV-LacZ had a dosedependent increase in LacZ reporter gene expression at each of the MOI'sused. The controls for each experiment were as follows: Mock (cellsalone, no treatment) and cells treated with each of the various UVdosages (500 J/m², 1000 J/m², 3000 J/m², 6000 J/m², which had RLU levelsconsistent with the Mock cultures (data not shown). Statisticalsignificance was calculated using the Student T-Test. The results areshown below in FIGS. 6-8.

Although this invention has been disclosed in the context of certainpreferred embodiments and an Example, it will be understood by thoseskilled in the art that the present invention extends beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses of the invention and obvious modifications thereof. It willbe appreciated, however, that no matter how detailed the foregoing mayappear in text, the invention may be practiced in many ways. Thus, it isintended that the scope of the present invention herein disclosed shouldnot be limited by the particular disclosed embodiments described above,but should be determined only by a fair reading of the claims thatfollow and any equivalents thereof.

1. A gene therapy system for increasing the transduction of anultraviolet light activated viral vector in a patient's spinal tissuecomprising: a power source; a light source producing an ultravioletlight beam, the light source being powered by the power source; a lightdelivery cable; an optical coupler for guiding the light beam into thelight delivery cable; a timed shutter in line with the light beam; auser interface for controlling the shutter; a light probe which receivesthe light beam from the light delivery cable, the light probe beingconfigured to output the light beam proximate to the target cells in apatient's spine in order to increase the transduction of the ultravioletlight activated viral vector; and an optical connector for connectingthe light delivery cable to the light probe.
 2. The system of claim 1,wherein the vector is recombinant adeno-associated virus (r-AAV).
 3. Thesystem of claim 1, wherein the light source is a laser.
 4. The system ofclaim 1, wherein the light source is a lamp.
 5. The system of claim 1,wherein the light source is configured to output light having awavelength from 255 nm to 400 nm.
 6. The system of claim 5, wherein thelight source is configured to output light having a wavelength of about325 nm.
 7. The system of claim 1, wherein the light source is configuredto output light having a wavelength from about 280 nm to about 330 nm.8. The system of claim 5, wherein the light source is a helium cadmiumlaser.
 9. The system of claim 1, further comprising a spinal implant towhich recombinant adeno-associated virus (r-AAV) is integrated.